PROJECT SUMMARY Much remains unknown about the network that controls the ubiquitin-dependent regulation of the most frequently inactivated protein in all human cancer: the tumor suppressor p53. A complete understanding remains elusive because current methods are unable to fully unravel how ubiquitin is led through the sequence of E1-E2-E3 enzymes to its final target. Mounting evidence suggests that E2s have a tremendous impact on both E3 ligase activity and the resulting ubiquitinated products. One gap in our knowledge is the identity of the E2 ubiquitin-conjugating enzyme (or enzymes) responsible for p53 ubiquitination in vivo. Given the combinatorial nature of interactions between ~40 human E2s and >600 E3s, this network is extraordinarily difficult to study. As a result, there is a striking need for tools that can follow ubiquitin through the enzymatic cascade to its target protein. The long-term goal of this proposal is to isolate and examine the network of protein-protein interactions that regulate p53 ubiquitination in living cells. This goal will be accomplished using a new method developed in the applicant?s lab, called targeted Charging of Ubiquitin to E2 (tCUbE), that can track a tagged ubiquitin as it moves from an E2 enzyme to its target protein. This strategy is unique in its ability to follow ubiquitin through the sequential E1-E2-E3 cascade to its ultimate target, and can therefore be used to provide a systems-level view of the ubiquitin-dependent regulation of p53. This application focuses on the questions surrounding the ubiquitin-dependent regulation of p53 activity. In Aim 1, tCUbE is used to discover the E2 network responsible for p53 ubiquitination in vivo. This method will identify the E2(s) that lead to p53 ubiquitination in vivo, and will enable experiments in which this network is perturbed using known molecules that inhibit p53 ubiquitination by the E3 ligases Mdm2 or MdmX. These studies will validate tCUbE, identify previously unknown interactions that guide p53 ubiquitination, and evaluate their impact on cancer phenotype. In Aim 2, tCUbE is used to interrogate a unique mode of p53 rescue mediated by an E2, UbcH7, which has been shown to protect p53 from degradation in certain cell types. Using tCUbE, it will be possible to evaluate the hypothesis that UbcH7 catalyzes the attachment different types of ubiquitin chains on substrates, like p53, that are protected from degradation. Finally, in Aim 3, tCUbE is combined with variants of ubiquitin that are resistant to deubiquitinase activity in order to profile the effects of deubiquitinases on the regulation of p53 function. These studies will test the hypothesis that distinct deubiquitinases disassemble specific ubiquitinated p53-species, and will evaluate how p53 activity can be differentially controlled through this mechanism. This approach is unmatched in its ability to illuminate the subset of ubiquitinated products that arise from the activity of a single E2 in living cells, thereby revealing the diverse network of interactions that mediate p53 ubiquitination. Such unique insight will enable the discovery of new potential targets for cancer therapy, and facilitate the development of new therapies that can disrupt specific steps within the ubiquitin path.